人源胰蛋白酶-1与DsbA在E.coli中的融合表达  

Fusion Expression of Human Trypsin-1 with DsbA in E. coli

作　　者：张文勇 王大梅 甘一如[1] 黄鹤[1] 

机构地区：[1]系统生物工程教育部重点实验室,天津大学化工学院,天津300072 [2]北京甘李药业有限公司,北京100026

出　　处：《化学工业与工程》2014年第6期75-79,共5页Chemical Industry and Engineering

基　　金：国家自然科学基金赞助项目(31470967);国家科技重大专项资助项目(2011ZX09201-301-05和2014ZX09508006-002-002)

摘　　要：从供体动物获取的胰蛋白酶可能含有致病微生物的残留,目前急需重组胰蛋白酶的生产。按照密码子使用频率表,对人源胰蛋白酶-1的基因序列进行简并突变,并与质粒pET-39b(+)携带的DsbA融合,实现了胰蛋白酶在大肠杆菌中的表达。重组蛋白主要以包涵体的形式存在,约占菌体总蛋白的40%。包涵体经洗涤、变性、复性和凝血酶激活后,依照市售猪胰蛋白酶的量,每1 L发酵液得到1.6 mg有活性的胰蛋白酶,实现了人源胰蛋白酶在原核生物中的大量表达。The trypsin obtained from animal sources may contain infectious agents,currently it is in urgent need of production of recombinant trypsin. According to the codon usage frequency table,the bases of human trypsin-1 were changed by degenerate codon,and then the sequences were fused with the DsbA fragment C-terminal in the vector pET-39b（ ＋）. The cloned E. coli with recombinant vector achieved expression. Recombinant proteins mainly existed in the form of inclusion bodies,accounting for about40% of the total bacteria proteins. By washing the pellet proteins,denaturation,renaturation and thrombin activation,we got 1. 6 mg activated trypsin per liter fermented liquid with the commercial porcine trypsin as a standard,and achieved efficient human trypsin expression in prokaryotes.

关 键 词：人源胰蛋白酶-1 包涵体 DSBA 

分 类 号：Q55[生物学—生物化学]